Safety:

Personal protective equipment consisting of a lab coat and nitrile gloves were worn throughout the duration of the experiment. Hands were washed with soap and water prior to and after the handling of fiddler crabs to reduce the transfer of bacteria.

Study Overview:

This study evaluates the extent to which increased air temperatures impact the escape speed of both male and female North American sand fiddler crab, Uca pugilator. Fiddler crab locomotion was observed under three different environmental temperature conditions: 24°C–26°C(Negative control), 29°C–31°C (Experimental Arm) , 32°C–34°C (Positive Control). The first temperature range was supported by Wilkens and Fingerman who categorized 24°C–26°C as the standard air temperature in marshland environments (1965). The additional two temperature ranges that were evaluated were selected based on Vianna’s (2020) study which demonstrated increased rates of burrow retreat when exposed to elevated temperatures of 29°C–31°C and 32°C–34°C. While these previous studies have demonstrated the effects of increased environmental temperature on fiddler crab behavior and physiological responses, this study looks to fill an existing knowledge gap of the effects of increased environmental temperatures specifically on locomotion. This investigation is prompted by evidence of climate change which underscores the potential threat to marshland ecosystems due to alterations in fiddler crabs' ability to escape from predators. I hypothesized that displacement would decrease as temperature increases as shown in the figure. 

Nine fiddler crab test subjects underwent the same testing under each of the three aforementioned environmental temperature ranges. A testing arena consisting of a sand-silt-mud mixture base and a burrow was warmed using heat lamps (Amazon#B07CXL3C6S) until the desired temperature was reached. In each arm prior to testing, the fiddler crab was warmed under a heat lamp (Amazon#B07CXL3C6S) to standard air temperatures before being transported to the testing arena. 

At the beginning of each trial, crabs were exposed to external stimuli to elicit a fear response and their locomotor movement, namely speed, was evaluated at both 5-seconds and 2-minutes after stimuli exposure for each trial. While these evaluation periods are significantly shorter than that of Allen et al.'s (2014), these adaptations were made to reflect the smaller scale at which this study is being conducted given the classroom setting. Preliminary trials indicated that 5-seconds was an acceptable time for evaluating immediate reaction to the external stimuli and 2-minutes for evaluating reaction over a longer period of time. 

Throughout the trial, qualitative data on crab health and behavior was recorded daily. Crab speed, movement, and color were all monitored. Additionally, quantitative air temperature and sand temperature data was recorded prior to each trial. For each experimental trial, crab locomotion was analyzed, and final quantitative crab displacement 5-seconds and 2-minutes after stimuli exposure were recorded.

Storage Terrarium Setup:

Storage terrarium setup was completed within the vicinity of a functional electrical outlet. The evening before the fiddler crabs kits were received, dechlorinated water was prepared by allowing a bucket (Amazon#B0032JUZ1K) of water to sit overnight with saran wrap sealing it, preventing evaporation.

Two 20 cm x 30 cm x 20 cm terrariums included in the Carolina Biological Fiddler Crab Kits (Carolina#142440) were prepared for storing fiddler crab test subjects – one terrarium for each kit of fiddler crabs. Each of these terrariums were labeled “Tera 1” and “Tera 2” by using a Sharpie and tape. The base of both terrariums was filled 5 cm deep using the sand from the 15 cm x 23 cm bag of sand included in the kits (Carolina#142440). A petri dish containing crab food, also included in the crab kits (Carolina#142440), was placed on one side of each terrarium. On the opposite side of each terrarium, a small plastic storage tank – included in the fiddler crab kit (Carolina#142440) – was positioned and filled with 400 mL of the prepared dechlorinated water, to which a pinch of salt was added to achieve the necessary salinity for crab's health and survival (Figure 1). This allowed each terrarium to maintain an aquatic environment on one side in contrast with the terrestrial environment on the other side. 

Lastly, a heating system was installed in each terrarium. This system was equipped with a Reptizoo Reptile Heat Lamp 100W bulb (Amazon#B07CXL3C6S) to maintain the terrarium temperatures of (24-25℃) and an outlet with a mechanical timer (Amazon#B00MWHQZX0) to prevent excessive heating.

Subjects and Care Notes: 

Water within each terrarium's storage container was replaced once per week using the dechlorinated water prepared as detailed in the Storage Tank Setup section above. Additionally, the saran wrap covering the 9L bucket (Amazon#B0032JUZ1K) of water was changed after each use. The sand-silt-mud substrate in both terrariums was hydrated regularly, and the temperature was carefully monitored using a REPTI ZOO terrarium thermometer (Chewy) to ensure a stable habitat temperature (24℃–28℃) was maintained throughout the study. Each of terrarium was regularly provided with 3 pellets of crab food from the kits (Carolina#142440) every 3 days for the duration of the experiment, and food was placed on a petri dish within the terrarium. 

Heat lamps (Amazon#B07CXL3C6S) were turned on for 15 minute increments every 3 hours to ensure storage terrarium air temperature was within a 24℃ to 28℃ range at all times. This temperature range is supported by previous research which has indicated that maintaining a temperature range of 24℃ to 28℃ is crucial to the health of crabs (Vianna et al., 2020). Additionally, given that fiddler crabs are ectotherms — an organism that relies on external processes to control its body temperature —, maintaining a stable temperature is key to their survival (Hews et al., 2021). 

A qualitative data collection table recorded in Google Sheets was used to document daily observations of fiddler crab behavior with “Day 0” representing the day crabs were received. In this table, frequency of watering and feeding behaviors were noted along with brackish water color. Between experimental trials, fiddler crabs were gently encouraged into 400 mL beakers and transferred to the appropriate terrarium. 

Test Subject Preparation:

Given natural death among the test subject population, nine crabs of the original twelve were selected. While this test population was significantly less than the 120 crabs Allen et al. (2014) evaluated in their experiment, given the classroom environment, this adjustment was made given storage capacity and to facilitate a replicable experiment in the time permitted. Waterproof acrylic pens (Amazon#B0C5MMYDRS) were used to paint unique colors on the shells of these nine crabs in order to identify subjects for data collection purposes. Crabs were carefully removed from their respective terrariums and held in a 400 mL beaker for 15 minutes, allowing them to air dry prior to being marked. Then, the shell of the crab was gently blotted with the tip of the pen. To ensure that the paint adhered to their shells, they were held in the 400 mL beaker for an additional 15 minutes allowing the ink to dry before being placed back in their respective terrarium. 

Testing Arena Setup:

All experimental trials were conducted in a Large 41 Quart Clear Plastic Storage Tank (Amazon#B07TTKCZRQ) labeled “Tera 3.” A meter stick was first placed along the length of the container's wall and secured using clear tape (Amazon#B0BMGGSTXX) to measure distance. A 66 cm by 27 cm section of the storage tank floor starting at 0 cm along the meter stick was covered with styrofoam squares (Amazon#B09V18ZH18) and approximately 3 cm deep of marine sand (Amazon#B0B52DC9VB) was added atop the styrofoam to create a uniform layer across this area. The styrofoam base was constructed in order to conserve the silt sand mud mixture while still providing a suitable surface layer for the fiddler crabs to move upon. 

Then, 1 L of marine sand (Amazon#B0B52DC9VB) and the entire 6 L bag of silt-sand-mud mixture (Gulf of Maine) were combined thoroughly in a 9 L bucket (Amazon#B0032JUZ1K) to closely resemble the fiddler crabs’ marshland ecosystem’s environment (Salimi et al., 2021). The remaining 18 cm by 27 cm section of the container was intentionally left without styrofoam to create a burrowing zone with sufficient depth of 7 cm (Figure 2). This section was filled with the substrate until the surface was level with the sand-styrofoam layer. Then, a circular hole measuring approximately 7 cm deep and 2 cm in diameter was manually formed at the 71 cm mark using a finger to simulate a natural burrow (Shinoda et al., 2019, p. 1). This setup was created in order to more accurately replicate fiddler crabs' natural environment (Shinoda et al., 2019, p. 1). 

For temperature control within the testing arena, three heat lamps (Amazon#B07CXL3C6S) were attached to stands and placed on both sides of the testing arena. One heat lamp was centered behind the wall with the meter stick attached, and the other two heat lamps were placed on opposite ends of the wall parallel. A REPTI ZOO terrarium thermometer (Chewy) was installed on the wall of the container roughly 8 cm above the surface of the substrate to monitor the air temperature.

A tripod with an iPhone was positioned at a high camera angle above the terrarium to capture an unobstructed view of the meter stick gradations and the full surface area of the testing arena. This camera angle was chosen to facilitate detailed observation of the crab’s behavior and shell color marking during experimentation while also providing a clear view of the meter stick markings to allow for accurate position measurements during testing. All trials were recorded using the iPhone similar to experiments by How et al. (2015) and Bagheri et al. (2022) who utilized digital recording methods to precisely capture and analyze the behavior of test subjects. These recordings allowed for an added retrospective trial analysis providing more detailed and precise observations of crab behavior and location in space compared to only real-time data recording.

Incorporating the evolutionary adaptations of fiddler crabs, particularly their unique polarized vision, was instrumental in simulating a realistic predator scenario for experimentation (How et al., 2015). Fiddler crabs have developed the ability to detect polarized light – a visual capability that significantly enhances their detection of predators through increased contrast in their environment (Bagheri et al., 2022). To leverage this adaptive trait, an iPad was held against the side of the testing arena to display a polarized image of a bird (Figure 3). This image was taken from How et al.'s research on fiddler crabs' use of polarized vision for target detection as shown in figure 3 (2015). Given that this image closely mimics a realistic scenario where fiddler crabs would use their polarized vision to detect predators, the image was used to elicit a fear response – running towards the burrow – in order to evaluate escape speed (How et al., 2015; Bagheri et al., 2022). 

In addition to the polarized image, a 500 g weight (Amazon) was attached to the end of a string. This external stimulus was implemented so that, when dropped from an 8 cm height behind the crab, it would further elicit an escape response given that fiddler crabs use visual cues such as expansion speed, angular size, and elevation above the horizon to determine the legitimacy of a threat (Hemmi, 2005, p. 1). The falling weight drew upon these visual cues leading the crab to exhibit fear and escape responses similar to those exhibited in a natural environment (Hemmi, 2005, p. 1). This is also similar to Allen et al.'s (2012) design in which crabs were physically chased down a lane to evaluate fiddler crab locomotion under heat stress (2012); the weight was an adaptation given the smaller scale of this experiment's testing arena. A photograph illustrating this setup is provided in Figure 4. 

Testing Arena Temperature:

While many procedural aspects of Allen et al.'s (2012) experimental design were similar, given the objective of evaluating locomotion, their experiment specifically focused on crabs whose internal temperatures were controlled. This experiment's procedure for controlling testing arena temperature follows that of Wilkens and Fingerman (1965). They used heat lamps to control environmental temperature. This experimental design focuses on ambient temperature variations, which are also relevant to the natural conditions experienced by fiddler crabs. Evidence of this is shown by their migratory responses to climate change (Johnson, 2014).

Across all arms of the study, prior to testing trials, heat lamps (Amazon#B07CXL3C6S) installed in the Testing Arena Setup section were used to control the temperature, and air temperature was monitored using the terrarium thermometer noted in the Testing Arena Setup section. Before testing could begin, the temperature was first measured to determine if it was in the specified range for each trial. If the temperature needed to be increased, the heat lamps were turned on longer, and the terrarium thermometer was monitored in 1-minute intervals. Once the temperature fell within the desired range, the heat lamps were turned off. If temperatures exceeded this accepted range, the heat lamps were turned off until the temperature reached the desired range.

Negative control trials were conducted within the standard marshland temperature range of 24-26°C to establish a baseline for fiddler crabs escape speed. In Wilkens and Fingerman's (1965) experiment, they evaluated the behavior and survival rates of 100 fiddler crabs under nine different temperature conditions, increasing in three-degree increments from 21°C to 45°C. This data led them to conclude that the optimal air temperature range for the North American fiddler crab: 24°C and 26°C.

For the positive control and experimental arm trials, a VIVOSUN Digital Thermostat (Amazon#B015F4VFGI) was installed to monitor the sand temperature. However, given that in preliminary trials the sand temperature was similar to that of the air temperature, sand temperature was not expected to be within a set temperature range. 

The positive control trials were conducted with an air temperature range of 32°C–34°C. In Allen et al.'s (2014) experiment, fiddler crabs exhibited extreme fatigue and slowest sprint speeds with an internal body temperature in this extreme range. In experimental trials, crabs were exposed to an experimental temperature range of 29°C–31°C to investigate their escape speed following stimuli exposure. This temperature was chosen, because it mimics the currently predicted increase in marshland temperatures in the northern hemisphere by the year 2100 (Vianna et al., 2020). Therefore, by gathering new data on the North American fiddler crab's speed and reaction time for a temperature range of 29°C–31°C, this study will aid in illuminating the impact of warming on marshlands.

Escape Speed Testing:

All trials across the positive control, negative control, and experimental arms followed a uniform procedure once the testing arena temperature stabilized within the desired range, ensuring consistency in experimental conditions. 

After one of the nine test subjects was selected randomly for testing, they were transported in a 400 mL beaker from their terrarium to an empty workstation. The beaker was placed under a heat lamp ((Amazon#B07CXL3C6S) for 10 minutes to ensure that their internal body temperature was within a normal range. This step was incorporated based on observations from preliminary testing in which fiddler crabs were moving very slowly and infrequently prior to testing. Additionally, it was observed that following exposure to the iPad and weight stimuli, crabs that were not warmed prior to testing did not exhibit sprinting behavior.  Their decreased locomotion was hypothesized to be attributed to a decreased internal body temperature. While the air temperature in the terrariums was within range, the water within the storage container in the terrariums was cold to the touch, leading to the conclusion that the crabs required an additional warming period to bring their internal temperature to an appropriate level (Weinstein & Full, 1994). Crabs were closely monitored while heating, and after 10 minutes, all test subjects exhibited increased movement and began to exhibit sprinting behavior during experimental trials.

After the preliminary warming period, the recording was started, and the test subject was then transferred to the testing arena. The crab was gently encouraged to enter the testing arena at a predetermined starting point at which the center of their shell was in line with the 8 cm mark on the meter stick. Then the 500 g (Amazon) weight was dropped from a height of 10 cm at the 2 cm meter stick marking to simulate a predator threat to elicit an escape response. These locations were designated to allow for ample space between the drop point of the weight and crabs to avoid risk of injuring the crabs. Immediately after the weight was dropped, the iPad with the polarized bird image from the Testing Arena Setup section was also displayed on the wall of the tank behind the crab's starting position to further mimic a predatory threat (Hemmi, 2005, p. 1).

Following stimuli exposure, crab behavior was recorded for a 2-minute period. This duration was determined based on prior preliminary trials where it was observed that fiddler crabs no longer exhibited new escape response behaviors after 2 minutes. Following each trial, a subsequent 10-minute rest period was given prior to repeating the trial for each crab. This rest period was supported by Allen et al. (2014) who similarly incorporated a 15-minute rest period for crabs between trials. Due to time constraints, preliminary tests were conducted evaluating crab behavior after shorter rest periods, and it was observed that a 10-minute rest period was just as effective in allowing crabs to recover as a 15-minute rest period.

Three sequential trials per each of the nine biological replicates were conducted for each arm, resulting in three data points for each test subject and 27 data points per arm of the experiment. 

Data Storage & Processing: 

Upon the completion of each trial, the video recording was immediately renamed to reflect the trial arm, test subject color identification, and trial number, adopting a structured format like "Negative Control, Dark Blue, Trial 1" for straightforward identification and ease of reference. The naming convention was designed to keep a chronological order of the trial number for each replicate, with sequential numbering indicating the sequence in which trials were conducted. This resulted in three trial recordings per replicate. These videos were then stored within folders titled corresponding to the arm: "Negative Control," "Positive Control," "Experimental." 

Once all trials were completed, trial recordings for each experimental arm were reviewed for each crab. The location of the fiddler crabs for both the 5-second and 2-minute data points was determined using the middle of the crab's shell as their location relative to the meter stick. Fiddler crab speed was calculated 5 seconds after the weight was dropped using the timestamps on trial recordings and the meter stick as reference for the crab’s exact location. Time was measured for each test subject and trial using timestamps from the recordings. Start time was defined as the time at which the weight was dropped near the crab. After 2 minutes from the exposure to stimuli, maximum displacement from the start point at 8 cm was calculated. This information was then recorded in the Google Sheet that was provided in the Data Collection procedure. 

After the data was entered for each arm of the experiment, an outlier analysis was conducted. This was done by calculating the mean displacement for each arm of the experiment at 5-seconds and 2-minutes after the stimuli response. For the negative control arm, an outlier was considered a crab that was +/- 20 cm from the mean. Any data point meeting this threshold was considered an outlier and eliminated from statistical analysis. The outlier was defined based on the 20 cm standard deviation from preliminary positive control data. Thus, data points that exceeded this amount were most likely to be outliers. For more detail on how to calculate an outlier this YouTube video was consulted.

A two tailed correlated t test was conducted between negative control and positive control trial data to evaluate the effects of  the experimental temperature range of (29°C–31°C)  on fiddler crab sprint speed over both 5 seconds and 2 minutes. For both analyses, the significance threshold was set at p < 0.05. A p-value less than 0.05 indicated a statistically significant difference in crab displacement due to temperature changes, supporting the hypothesis that temperature affects crab mobility. Conversely, a p-value greater than 0.05 would suggest that the temperature variations do not significantly alter the crabs' displacement, thereby supporting the null hypothesis.